The effects of resveratrol in animal models of primary osteoporosis: a systematic review and meta-analysis

Background There is still a lack of sufficient evidence-based medical data on the effect of resveratrol (Res) on primary osteoporosis (OP). This meta-analysis aimed to comprehensively evaluate the role of Res in animal models of primary OP. Methods The PubMed, Cochrane Library, Web of Science and Embase databases were searched up to August 2023. The risk of bias was assessed by the SYRCLE RoB tool. Random- or fixed-effects models were used to determine the 90% confidence interval (CI) or standardized mean difference (SMD). Statistical analysis was performed with RevMan 5.4 and Stata 14.0. Results A total of 24 studies containing 714 individuals were included. Compared with those in the control group, the bone mineral density (BMD) (P < 0.00001), bone volume/total volume (BV/TV) (P < 0.001), trabecular thickness (Tb.Th) (P < 0.00001), and trabecular number (Tb.N) (P < 0.00001) were markedly greater, and the trabecular separation (Tb.Sp) (P < 0.00001) was significantly greater. Compared with the control group, the Res group also exhibited marked decreases in alkaline phosphatase (ALP) (P < 0.05), tartrate-resistant acid phosphatase 5b (TRAP5b) (P < 0.01), and type I collagen strong carboxyl peptide (CTX-1) (P < 0.00001) and a marked increase in osteoprotegerin (OPG) (P < 0.00001). Conclusion In summary, we concluded that Res can markedly increase BMD, improve morphometric indices of trabecular microstructure and serum bone turnover markers (BTMs), and exert a protective effect in animal models of primary osteoporosis. This study can supply experimental reference for Res in primary osteoporosis treatment. Supplementary Information The online version contains supplementary material available at 10.1186/s13018-024-04595-1.


Introduction
Osteoporosis (OP) is a systemic bone disease characterized by damage to the bone microstructure and decreased bone mass, resulting in bone fragility and easy fracture [1,2].Primary osteoporosis, as a major part of OP, is currently a major public health problem facing patients and medical practitioners globally.A decreasing BMD not only increases the incidence of fractures but also has an incalculable impact on patients' financial status and personal and even whole-family quality of life, given that most patients with primary OP are elderly patients (postmenopausal OP and senile OP) [3,4].Clinically, anti-OP drugs are categorized into anti-absorptive and pro-synthetic drugs.Widely used options include bisphosphonates (BP), selective estrogen receptor modulators (SERM), and RANK-ligand inhibitors.Despite their popularity, these drugs are associated with various adverse effects.For example, Denosumab, a type of BP, significantly increases BMD in the spine and hip of postmenopausal women with OP.However, due to severe gastrointestinal reactions such as acid reflux, nausea, and vomiting, many patients opt for intravenous administration over oral, potentially impacting treatment compliance of patients [5][6][7][8].For another example, although oestrogen replacement treatment has a significant effect on treating postmenopausal OP [9], studies have shown that this therapy may increase the risk of breast and uterine cancer.Thus, there is a critical need to identify drugs that are more effective, convenient, and safer for primary OP.
Resveratrol (Res) is a polyphenolic phytoestrogen that is present in the skin of red grapes, peanuts and various other fruits [10,11] and has potent antioxidant, antiinflammatory, antiageing, neuroprotective, anticarcinogenic and cardioprotective effects [12,13].In vitro evidence has shown that Res can improve the activity of osteoblasts and inhibit the differentiation of osteoclasts [14,15].For example, in vitro, Res increases ALP in a dose-dependent manner by promoting the differentiation of osteoblasts [16].In vivo studies have shown that Res can improve BMD and prevent bone loss in young rats subjected to tail suspension, in ovariectomized (OVX) rats and in old rats subjected to hind limb suspension [17][18][19].However, a large number of existing studies have not yet systematically summarized and analyzed the topic.Therefore, this review aims to comprehensively explore the role of Res in an animal model of primary OP through the use of a meta-analysis of animal experiments for the first time.

Methods
The meta-analysis was performed in accordance with PRISMA guidelines (Additional file 1 ) and registered on the PROSPERO platform of the International Register of Systematic Evaluations (No. CRD42023478041).

Data sources and search
Cochrane Library, PubMed, Web of Science and EMBASE were searched for studies of Res in animal models of primary OP up to August 2023.

Study selection
The inclusion criteria were as follows: (1) The animal model was primary OP; (2) The animal models of primary OP were established by all kinds of methods, such as age-related OP, orchiectomy and ovariectomy; (3) The treatment group was given Res only, while the control group was given either no treatment or saline treatment; (4) The main results were bone mineral density (BMD); the second outcomes were morphometric indices of trabecular microstructure, including bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp); and serum bone turnover markers (BTMs), including osteocalcin (OC), alkaline phosphatase (ALP), serum osteoprotegerin (OPG), bone alkaline phosphatase (bALP), type I collagen strong carboxyl peptide (CTX-1), and tartrateresistant acid phosphatase 5b (TRAP5b).The exclusion criteria were as follows: (1) reviews, cases, clinical trials, cell studies or other studies; (2) other animal models; and (3) other medicines.

Data extraction
Two authors independently extracted the study characteristics (publication year, first author and sample size), method of modeling, basic characteristics, intervention, and outcome information.All the data were acquired, and several subgroup analyses were carried out for different dosages, modeling-established standards or patient positions.Disputes between the two radiologists were resolved by talking with a third person.

The risk of bias assessment
The risk of bias of the included studies was evaluated by the SYRCLE risk of bias tool [20] (Fig. 2); the risk of bias was classified as "high", "low" or "unclear".Disagreements between An and Luo were resolved by Dr. Jin.

Subgroup and sensitivity analysis
Because of the limited sample size, the number of female animals was greater than the number of male animals, the data on age and weight were incomplete, and the methods of modeling were different.In this review, a subgroup analysis was conducted even though it was difficult.If there was obvious heterogeneity in the primary outcome (I 2 > 50%), this study was subjected to sensitivity analysis.Moreover, the stability of all outcomes was evaluated by ignoring each study in sequence.

Data synthesis
Excel 2016, Stata 14.0, and RevMan 5.4 were used to perform this analysis.When the data were reported as the mean ± SEM (standard error of the mean), we transformed the SEM into the standard deviation (SD) using the formula ðSEM = SD/square root of the sample sizeÞ to avoid obfuscating the distinctive usage between the SD and SEM.Statistical heterogeneity was assessed by the chi-square test and the I 2 test.A fixed-effects model was selected if I 2 was < 50%; otherwise, the random-effects model was selected.Several independent groups in a study (e.g., various doses) were considered separate datasets.P < 0.05 indicated statistical significance.

Results
A total of 714 studies were selected.After removing duplicates, 351 studies remained.Sixty studies were left for full-text screening after screening the titles and abstracts.Finally, 24 studies were analyzed.The basic characteristics of the final 24 studies [15,17,19, are shown in Table 1.The search process is shown in Fig. 1.

The risk of bias and publication bias
Several studies (Fig. 2) were thought to have an "unclear risk of bias", for example, random sequence generation, random housing and random outcome assessment.A low risk of bias was observed for incomplete outcome data, baseline characteristics and selective reporting in all studies except one [19].Moreover, the funnel plot (n > ten papers) showed that the stability of the results was not affected by publication bias (Additional file 1 ).

Effectiveness
Primary outcomes-BMD (Figs. 3 and 4) Analysis of 45 studies [17, 19, 23-26, 28-32, 34, 36, 39, 40] showed that, compared with the control group, the Res group had a markedly greater BMD (n = 587; SMD, 1.59; 95% confidence interval (CI), 1.22 to 1.96; I 2 = 60%, P < 0.00001).Due to the high heterogeneity, we analyzed the BMD subgroups according to the methods of modeling, test methods and test positions (Fig. 4).Subgroup analysis according to the above several points showed no significant reduction in heterogeneity, which may remind us to search for other more suitable points (Additional file 1).

Discussion
This review assessed the protective effects of Res in animal models of primary osteoporosis.Twenty-four articles were analyzed, and eleven results were obtained.This review showed that Res can markedly increase BMD,    improve morphometric indices of the trabecular microstructure and serum BTMs concentration, and exert a protective effect on animal models of primary OP.BMD, a gold standard for diagnosing OP, can be detected via dual-energy X-ray (DXA) or micro-CT.Mizutani K et al. reported that Res can alleviate the decrease in femoral BMD induced by ovariectomy in rats [18,42], while Li YT et al. suggested that inhibiting bone resorption may be related to the ability of Res to increase BMD because Res can inhibit the production of prostaglandin e2 and interleukin-6 [43].Kenny reported that the serum testosterone concentration is positively correlated with the BMD, and Res may ameliorate bone loss caused by male hypogonadism by maintaining the balance between RANK and OPG [31].Therefore, our study evaluated the ability of Res to improve BMD in primary OP patients.This study showed that the BMD in the Res group increased significantly.In addition, the subgroup analysis according to the modeling methods, detection methods or detection positions also yielded significant results.In addition, due to the high heritability of BMD, one study had explored the reason why individual differences exist in the effectiveness of bisphosphonates (a first-line anti-OP drug at present) from the genetics.The findings revealed that, in contrast to rs1544410 A/G, another variant, rs2228570 C/T associated with the vitamin D receptor, exhibited a correlation with a favorable response to antiresorptive therapy.This study prompts us to explore Fig. 5 The meta-analysis results of Res for BV/TV Fig. 6 The meta-analysis results of Res for Tb.N whether the genetic characteristics of diverse primary OP conditions influence the efficacy of resveratrol in improving BMD, given its promising results [44].
It is well-known that the morphometric indices of the trabecular microstructure play a vital role in the diagnosis of osteoporosis [45].The BV/TV is one of the key indices of trabecular microstructure, and an increase in this parameter indicates that bone anabolism is more common than catabolism and that the bone mass is greater, and vice versa.Ozturk S et al. reported that Res (80 mg/kg/day) can reduce the BV/TV, Tb.N and Tb.Th and prevent a sharp decrease in bone mass caused by ovariectomy by improving the microstructure and biophysical and chemical properties of bone [27].Therefore, our study evaluated the BV/TV in an animal model of primary OP and revealed that Res can improve bone mass and bone metabolism by increasing the BV/TV.In addition, Tb.Th, Tb.N, and Tb.Sp are the primary parameters used to evaluate the spatial morphological structure of trabecular bone.Once osteoporosis occurs, the Tb.Sp increases, while the Tb.N and Tb.Th decrease.Therefore, by evaluating the above three indices, this study revealed that Res can increase the Tb.Th and Tb.N while reducing the Tb.Sp, thus improving bone loss in an animal model of OP.Intermediate metabolites or enzymes produced during bone turnover are called serum BTMs.BTMs can be classified as bone formation or bone resorption markers.The former indicates osteoblast activity and bone formation, such as ALP, bALP and OC, while the latter reflects osteoclast activity and bone resorption, such as CTX-1 and TRAP5b.BTMs play a role in diagnosing various bone diseases, determining bone turnover types, predicting the risk of fracture, monitoring treatment compliance and evaluating drug efficacy [22,36,40].The level of BTMs in primary OP is usually normal or slightly elevated.Feng J et al. reported that Res inhibits the generation of osteoclasts in OVX rats by decreasing RANKL and TRAP5b and increasing OPG.This difference may be related to the antiapoptotic, antioxidative and antiinflammatory effects of Res [26].Elesawy reported that chronic administration of Res can significantly improve the BMD of the tibia, and the protective mechanism Fig. 9 The meta-analysis results of Res for ALP Fig. 10 The meta-analysis results of Res for bALP Fig. 11 The meta-analysis results of Res for OC may involve increasing the levels of OC, OPG and ALP [22].Therefore, the present study showed that ALP, CTX-1 and TRAP5b in the Res group were significantly decreased, and OC tended to decrease.In addition, bALP showed an opposite trend to that of the other indicators, which may be due to the small amount of data.OPG is a metabolite secreted by osteoblasts and can inhibit the formation of osteoclasts by competitively binding with RANK [22,36,40].Our results showed that the OPG in the Res group increased significantly, which improved the state of primary OP.In addition to the above markers, there are other bone metabolic intermediates, including p1np, p1cp, LCa/Cr, dpyr, and ntx [45,46].Unfortunately, this review did not pursue further investigation due to challenges in acquiring adequate data.
The study limitations were as follows: (1) Language bias; (2) the reliability of the risk of bias assessment was limited to low-quality included studies; and (3) most of the results were highly heterogeneous; however, we performed a subgroup analysis.The results of the sensitivity analysis were robust (Additional file 1).
Fig. 12 The meta-analysis results of Res for OPG Fig. 13 The meta-analysis results of Res for CTX-1 Fig. 14 The meta-analysis results of Res for TRAP5b

Conclusions
Res can markedly increase BMD, improve morphometric indices of the trabecular microstructure and serum BTM concentration, and exert a protective effect on animal models of primary osteoporosis.This study can provide an experimental reference for Res in primary OP.In the future, additional studies are needed to evaluate the effects of Res as an anti-primary OP drug.

Fig. 1 Fig. 2 Fig. 3
Fig. 1 Flowchart of the literature search and selection process

Fig. 4
Fig. 4 Subgroup analysis of Res for BMD

Fig. 7
Fig.7The meta-analysis results of Res for Tb.Th

Fig. 8
Fig.8The meta-analysis results of Res for Tb.Sp

Table 1
Characteristics of the included studies in the meta-analysis